The present invention relates to apparatus and methods for non-invasive techniques to detect diseases and for drug testing, to Raman spectroscopy and surface enhanced Raman spectroscopy.
Normal Raman spectroscopy relates to the scattering of light from a gas, liquid or solid with a shift in wavelength from that of the usually monochromatic incident radiation. Upon irradiation of a molecule with light in biological applications, the incident radiation having a frequency .nu. should produce scattered radiation, the most intense part of which has unchanged frequency. In addition, if the polarization of a molecule changes as it rotates or vibrates, there are spectral lines of much lesser intensity at frequencies .nu..+-..nu..sub.k, where .nu..sub.k is the molecular frequency of rotation or vibration.
Laser-induced fluorescence techniques have been used for cancer diagnosis. Normal Raman spectroscopy (NRS) also has been suggested as an alternate biomedical diagnostic tool since the Raman signals can provide complementary spectral information. However, Raman spectroscopy has several limitations. Intensity of the Raman signal is intrinsically weak and interference from the fluorescence signal is a major problem for the weak signals. In addition weak Raman signals often require sophisticated Fourier transform processing which requires relatively expensive equipment. The requirement of high-power lasers, present potential hazards for in vivo measurements, even with Fourier transform processing.
Fleischmann et al. first reported strongly enhanced Raman scattering from pyridine molecules adsorbed on silver electrode surfaces that had been roughened electrochemically by oxidation-reduction cycles (Chem. Phys. Lett. 26, 163, 1974). This increase in Raman signal, originally attributed to a high surface density produced by the roughening of the surface of electrodes, was later identified by Jeanmaire and Van Duyne (J. Electroanal. Chem. 84, 1, 1977) and independently by Albrecht and Creighton (J. Am. Chem. Soc. 99, 5215, 1977) as a direct result of a surface enhancement process, hence the term surface-enhanced Raman scattering (SERS) effect. In spite of extensive basic research in the 1970's, SERS started to become a useful and practical analytical technique only in the mid-1980's. Vo-Dinh and coworkers first reported the general applicability of SERS as an analytical technique for a variety of chemicals including several homocyclic and heterocyclic polyaromatic compounds on cellulose-based substrates covered with silver-coated microspheres, (T. Vo-Dinh, Surface-Enhanced Raman Spectroscopy, in Chemical Analysis of Polycyclic Aromatic Compounds, Wiley, New York, 1989; and T. Vo-Dinh, Surface-Enhanced Raman Spectroscopy, in Photonic Probes of Surfaces, Elsevier, New York, 1995). The origin of the enormous Raman enhancement appears to come from the results of several electromagnetic and chemical mechanisms. The observed Raman scattering signals for the adsorbed molecules were found to be more than a million times larger than those expected from gas phase molecules or from non-adsorbed compounds. These enormous enhancement factors, which help compensate for the normally weak Raman scattering process, open new horizons to the Raman technique.
Surface-enhanced Raman scattering (SERS) techniques enhance the Raman signal up to 10.sup.8 fold. Extensive efforts have been devoted to determining and investigating sources of that enhancement. There are at least two major types of mechanisms that contribute to the SERS effect: a) an electromagnetic effect associated with large local fields caused by electromagnetic resonances occurring near metal surface structures, and b) a chemical effect involving a scattering process associated with chemical interactions between the molecule ans the metal surface.
Although SERS techniques have been used for biochemical analysis, there has been no previous works on SERS-based techniques and instrument for in situ and in vivo biomedical diagnosis and drug testing. Perhaps this is because there are several problems associated with the use of SERS in biomedical applications. Until now, the SERS approaches, which use electrode systems or silver sol media, involve only in vitro samples and do not allow in vivo analysis.